Bone formation in vertebrates is a dynamic process involving continuous production of bone and continuous bone resorption. Osteopenia is a general term used to describe any bone-wasting disease in which the rate of bone resorption is greater than the rate of bone formation. Osteoporosis results from a progressive net loss of skeletal bone mass due to an increase in bone resorption exceeding bone formation. Osteoporosis afflicts nearly 20 million people in the United States alone, and total costs from osteoporosis-related injuries amount to at least $7 billion annually (Barnes, Science 236:914 (1987)). A major difficulty in monitoring the disease is the lack of a specific assay to measure acute changes which may occur from various treatment regimens. There is a need to have an easy, reliable test for bone formation.
Type I Collagen is unique to connective tissues and is a major component in bone, among other tissues. The normal synthesis and breakdown of this collagen type can be altered during the pathogenesis of many kinds of disease, including osteoporosis. Because bone is a metabolically active tissue throughout life, indicators of Type I collagen turnover could be useful as a markers in metabolic bone disease. However, the major means for estimating the metabolic rate of bone collagen has been to quantify the urinary excretion of hydroxyproline, which is derived from collagenous proteins. This test has proven tedious, associated with several sources of error, and not specific for Type I collagen. Azria, Calcif. Tissue Int. 45:7-11 ( 989).
Each Type I collagen fiber is composed of three long, helical polypeptide chains (.alpha. chains) that bind tightly to each other. Each Type I collagen polypeptide is synthesized as a larger procollagen molecule, containing additional sequences at both the amino and carboxy termini. It appears that the large amino and carboxy terminal ends of the procollagen are important in the alignment and binding of the trimer. The amino and carboxy terminal propeptides are cleaved extracellularly by specific proteinases before the .alpha. chains are assembled into fibers.
The portion of the procollagen polypeptide removed from the carboxy terminus, referred to as the carboxyterminal propeptide of Type I collagen, has been found in blood, where its concentration changes during growth and in metabolic bone disorders. Radioimmunoassays for the carboxyterminal propeptide have been reported (e.g., Taubman et al., Science 186:1115-1117 (1974); Taubman et al., Proc. Soc. Exp. Biol. Med. 152:284-287 (1976); and Parfitt et al. J. Bone Min. Res. 2:427-436 (1987)), including the use of digests of carboxy terminal procollagen obtained from cultured human skin fibroblasts and then purified by digestion, lectin affinity chromatography, gel filtration and ion exchange HPLC (e.g., Meikko et al., Clin. Chem. 36:1328-1332 (1990)). These tests, however, are difficult and expensive to prepare and have not found widespread use.
Little work has been reported with the amino terminal propeptide of Type I collagen. In 1987 it was reported that, in a group of patients with primary biliary cirrhosis, both Type I and Type III procollagen aminopropeptides were measured. This was performed by ELISA using purified antigen from calf skin. This study found that the amount of Type I amino propeptide was unchanged, regardless of the degree of histologic fibrosis on liver biopsy, whereas Type III procollagen varied significantly with disease state (Davis et al., Am. J. Path., 128:265 (1987)).
Accordingly, there remains a need for a sensitive and specific measure of bone formation which is practical to produce and convenient to use. The digests of native procollagen, for example, are not conveniently prepared, do not allow for the production of antibodies to well-defined epitopes of interest, and are expensive to develop. The present invention circumvents many of these problems while meeting the requirements for sensitivity and specificity, and fulfills other related needs.